Methods of Determining Efficacy of Therapy For Niemann-Pick C Disease And Related Disorders

ABSTRACT

A method for identifying and screening subjects for cholesterol storage or trafficking diseases using an oxysterol as a biomarker. More particularly, subjects are screened and can be identified as having Niemann-Pick C disease, the method comprising the steps quantifying the concentration of an oxysterol in a biological sample taken from the subject and comparing the concentration of the oxysterol of the subject to a reference value of the oxysterol derived from a non-affected subject population. If the concentration of the oxysterol from the subject is higher than the reference value, the subject is identified as affected with Niemann-Pick C disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 13/786,757 filed on Mar. 6, 2013, which is a continuation ofU.S. Non-Provisional application Ser. No. 12/385,529, now U.S. Pat. No.8,497,122, filed on Apr. 10, 2009, which claims the benefit of U.S.Provisional Application No. 61/071,074, filed on Apr. 11, 2008. Thedisclosures of these applications are incorporated herein by referenceeach in its entirety.

GOVERNMENT RIGHTS

This disclosure was made with government support under Grant No. P50HL083762 from the National Institutes of Health. The Government hascertain rights in the invention.

FIELD

The present disclosure relates to biomarkers for disorders involvingaccumulation of one of more oxysterols such as cytotoxic oxysterolaccumulation, Niemann-Pick C (NPC) disease, lysosomal storage diseases,cholesterol trafficking diseases, and neurodegenerative diseases. Thepresent disclosure relates to methods for diagnosing and screeningsubjects having lysosomal trafficking diseases relating to cholesteroltransport. In particular, the present invention relates to diagnosingand monitoring subjects having Niemann-Pick C disease or acholesterol-related neurodegenerative disorder by quantitativelymeasuring disease-specific plasma or cell membrane biomarkers.

BACKGROUND

The statements in this section merely provide background informationrelated to the present disclosure and may not constitute prior art.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomalstorage and neurodegenerative disorder. It can involve accumulation ofcholesterol and other lipids in the viscera and the central nervoussystem, and patterned Purkinje cell death in the cerebellum. NPC isdescribed on the On-Line Mendelian Inheritance in Man (“OMIM”) website,OMIM number 257220. NPC presents a highly variable clinical phenotype.In childhood-onset NPC, the patients typically appear normal for 1 or 2years with neurological symptoms, such as ataxia, grand mal seizures,loss of previously learned speech, spasticity, and seizures, appearingat 2 to 4 years. There are also prenatal and adult-onset forms of thedisease.

In NPC disease, two genetic complementation groups, NPC1 and NPC2, havebeen identified. Mutations in the NPC1 gene cause ˜95% of the cases, therest being caused by NPC2 mutations. Loss-of-function of the NPC1 genein mice yields marked impairment in both esterification of low-densitylipoprotein (LDL) cholesterol and mobilization of newly hydrolyzed LDLcholesterol to the plasma membrane, resulting in lysosomal sequestrationof LDL cholesterol, delayed down-regulation of the LDL receptor and denovo cholesterol biosynthesis, and impaired ATP-binding cassettetransporter (ABCA1)-mediated cholesterol efflux. Associated with theselipid trafficking defects, NPC1 mutants exhibit cellular oxidativestress, leading to increased production of non-enzymatic cholesterolauto-oxidation products.

Information regarding the biochemical and histopatholgical defectsassociated with NPC has come through the use of two murine models whichshare many of the clinical abnormalities observed in humans with NPC:elevated levels of sphingomyelin and unesterified cholesterol in liverand spleen, presence of foamy macrophages, neuronal vacuoles, focalaxonal swelling, and decreased Purkinje cell number. The two murine NPCmodels, C57B1Ks/J spm and BALB/c npc^(nih), arose as spontaneousmutations, were determined allelic by cross breeding, and have beenindependently localized to mouse chromosome 18 in a region syntenic tothe human NPC1 locus. Confirmation that the two mouse loci belong to thesame complementation group as the human NPC1 locus was determined usingheterokaryon fusions of human NPC1 fibroblasts to mouse mutant celllines and by DNA-mediated complementation using a yeast artificialchromosome (YAC) from the human NPC1 region. Combined, these studiesindicate that the same gene is altered in the two mouse NPC models (spmand npcnih) and that the orthologous gene in the mouse models isdefective at the human NPC1 locus.

Despite recent progress in characterizing the biochemical and geneticdefects in NPC disease, the mean time to diagnosis from initialpresentation is approximately five years. The delay in diagnosis islargely due to the lack of both newborn screening and diseasebiomarkers, as well as the lack of widely available diagnostic tests. Inaddition, the absence of biomarkers that correlate with disease severityhas hampered evaluation of the efficacy of therapeutic approaches to NPCdisease.

DRAWINGS

The drawings described herein are for illustration purposes only and arenot intended to limit the scope of the present disclosure in any way.

FIG. 1A shows the chemical structure of 24(S) hydroxycholesterol (hereinreferred to as “24HC”)

FIG. 1B shows the chemical structure of 7-ketocholesterol (hereinreferred to as “7-keto”).

FIG. 1C shows the chemical structure of cholesten-3β, 5α, 6β-triol(herein referred to as “triol”).

FIG. 2A-D show gas chromatograms/mass spectra (GC/MS) of oxysterols inplasma samples obtained from confirmed NPC patients and control non-NPCafflicted subjects. The arrows indicate oxysterol biomarkeridentification. The Y-axis is the measure of relative abundance ofsampled oxysterols measured against a known internal standard from whicha calculation of the concentration of the oxysterol in the biologicalsample can be made. The X-axis is the retention time of each of the ionspecies eluting from the GC column in minutes. The table below thecontrol and NPC samples summarizes the relative abundance of eachoxysterol measured using the identification and testing methods of thepresent disclosure in the NPC disease group and the control group.

FIG. 3A shows a graph depicting plasma levels of the oxysterol 7-ketoobtained from control non-afflicted subjects (“CONTROL”), confirmed NPCsubjects (“NPC”) and heterozygote but not NPC afflicted subjects havinga NPC1^(+/−) genotype (“HET”). The concentration of the oxysterol 7-ketois expressed as nanograms per milliliter (ng/mL) of plasma.

FIG. 38 shows a graph depicting plasma levels of the oxysterol triolobtained from control non-afflicted subjects (“CONTROL”), confirmed NPCsubjects (“NPC”) and obligate or confirmed heterozygote non-NPCafflicted subjects having a NPC1^(+/−) genotype (“HET”). Theconcentration of the oxysterol triol is expressed as nanograms permilliliter (ng/mL) of plasma.

FIG. 4 shows a graph depicting levels of the oxysterol triol fromcerebrospinal fluid (CSF) samples obtained from control non-afflictedsubjects (“CONTROL”) and confirmed NPC subjects (“NPC”). Theconcentration of the oxysterol triol is expressed as nanograms permilliliter (ng/ml) of plasma.

FIG. 5 shows the Pearson Correlation coefficient for the correlationbetween disease severity and plasma levels of 24-HC.

FIG. 6A shows a bar graph depicting the concentration of triolnormalized to the concentration of triol from NPC plasma samples treatedwith butylated hydroxytoluene (BHT). Treatments include: NPC plasmasamples treated with BHT (“BHT”), no BHT (“No BHT”), at 4° C. with BHT(“4 BHT”), at 4° C. without BHT (“4 No BHT”), at room temperature(20-21° C.) with BIT (“RT BHT”) and at room temperature (20-21° C.)without BHT (“RT No BHT”).

FIG. 6B shows a bar graph depicting the relative concentration of 7-ketonormalized to the concentration of 7-keto from NPC plasma samplestreated with butylated hydroxytoluene (BHT). Treatments include: NPCplasma samples treated with BHT (“BHT”), no BHT (“No BHT”), at 4° C.with BHT (“4 BHT”), at 4° C. without BHT (“4 No BHT”), at roomtemperature (20-21° C.) with BHT (“RT BHT”) and at room temperature(20-21° C.) without BHT (“RT No BHT”).

FIG. 7A shows a bar graph depicting the relative amount of triol fromNPC subjects normalized to amounts of triol in plasma samples obtainedbetween the hours of 7:30 am-8:00 am (“0730-0800”). Other timedintervals include between 4:00 pm and 5:30 (“1600-1730”) and between11:00 pm and 11:30 pm (“2300-2330”). Results are indicative of averagedvalues over 5 different measurements for each time point.

FIG. 7B shows a bar graph depicting the relative amount of triol fromNPC subjects normalized to amounts of triol in plasma samples obtainedbetween the hours of 7:30 am-8:00 am (“0730-0800”). Other timedintervals include between 4:00 pm and 5:30 (“1600-1730”) and between11:00 pm and 11:30 pm (“2300-2330”). Results are indicative of averagedvalues over 5 different measurements for each time point.

FIG. 8A shows a graph depicting plasma levels of the oxysterol 7-ketoobtained from control non-afflicted subjects (“CONTROL”), confirmed NPCsubjects (“NPC”) and from subjects having other lysosomal storagediseases, including those with known CNS involvement: infantile neuronalceroid lipofuscinosis (“INCL”). GM1 gangliosidosis (“GM-1”), GM-2gangliosidosis (“GM-2”) (Tay-Sachs Disease), Gaucher's disease (“GD”)and hepatosplenomegaly (“HSM”). The concentration of the oxysterol7-keto and triol are expressed as nanograms per milliliter (ng/mL) ofplasma.

FIG. 8B shows a graph depicting plasma levels of the oxysterol triolobtained from control non-afflicted subjects (“CONTROL”), confirmed NPCsubjects (“NPC”) and from subjects having other lysosomal storagediseases, including those with known CNS involvement: infantile neuronalceroid lipofuscinosis (“INCL”), GM1 gangliosidosis (“GM-1”), GM-2gangliosidosis (“GM-2”) (Tay-Sachs Disease), Gaucher's disease (“GD”)and hepatosplenomegaly (“HSM”). The concentration of the oxysterol triolis expressed as nanograms per milliliter (ng/mL) of plasma.

FIGS. 9A-H show bar graphs of oxysterols 24-hydroxycholesterol(“24-HC”), 25-hydroxycholesterol (“25-HC”), 27-hydroxycholesterol(“27-HC”), cholestane-3β,5α,6β-triol (“triol”) 4β-hydroxycholesterol(“4β-HC”), 7α-hydroxycholesterol (“7α-HC”), 7β-hydroxycholesterol(“7β-HC”), and 7-ketocholesterol (“7-keto”) isolated from wild-type andNPC1−/− null mice (NPC afflicted mice) as a function of age (weeks).Methods for isolating, identifying and quantifying the variousoxysterols were performed in accordance with the methods of the presentdisclosure.

SUMMARY

The present disclosure relates to the finding that one or more of a setof oxysterols may be used as a biomarker for screening, diagnosing, ormonitoring disorders involving accumulation of one or more oxysterolssuch as cytotoxic oxysterol accumulation, Niemann-Pick C (NPC) disease,lysosomal storage disease, and neurodegenerative disease. Oxysterols arecommonly referred to as oxidized derivatives of cholesterol, typicallygenerated non-enzymatically, enzymatically as a normal part ofcholesterol metabolism, or absorbed through dietary intake. Oxysterolstypically have a hydroxyl-, epoxy- or a keto-group on the cholesterolmolecule. In some embodiments of the present disclosure, the oxysterolcan be any one or more of 24(S)-hydroxycholesterol (“24-HC”),25-hydroxycholesterol (25-HC), 7-ketocholesterol (“7-keto”) andcholesten-3β, 5α, 6β-triol (“triol”).

An oxysterol may be present in a subject at a level that is elevatedcompated to the level of oxysterol in a population not afflicted with adisorder involving accumulation of one or more oxysterols such ascytotoxic oxysterol accumulation, Niemann-Pick C (NPC) disease,lysosomal storage disease, and neurodegenerative disease. Certainoxysterols can be present at levels below those found in controlpopulations, and/or at levels that vary over time. Levels of oxysterolscan be used for screening, diagnosing, and/or monitoring disordersinvolving accumulation of one or more oxysterols such as cytotoxicoxysterol accumulation, Niemann-Pick C (NPC) disease, lysosomal storagedisease, and neurodegenerative disease.

The present disclosure provides methods for identifying a subjecthaving, or at risk for developing, NPC disease by detecting an increasedconcentration of one or more oxysterols in a biological sample collectedfrom the subject. The present disclosure In some embodiments, thepresent disclosure provides a method for identifying a subject with NPCdisease that is characterized by an increased concentration of one ormore oxysterols in a biological sample collected from the subject.According to this method, steps for identifying a subject having NPCdisease comprises: (a) obtaining a biological sample from the subject;(b) quantifying the concentration of oxysterols comprising24-hydroxycholesterol, 7-ketocholesterol, cholesten-3β, 5α, 6β-triol orcombinations thereof in the biological sample; and (c) comparing theconcentration of the oxysterol present in the biological sample to areference value of the oxysterol obtained from a control population,wherein if the concentration of the oxysterol from the subject is higherthan the reference value, the subject is identified as affected with NPCdisease.

As used in the various methods for identifying and screening subjectshaving NPC disease, the biological sample obtained from the subject caninclude one or more of a blood sample (including one or more of wholeblood, sera, plasma, red blood cells or cord blood), a tissue biopsy(including one or more of single cells, or cultured cells from a tissuebiopsy), a cerebrospinal fluid sample, a sputum sample and an amnioticfluid sample.

In some embodiments, the methods described herein can be carried out toscreen subjects for lysosomal cholesterol storage and traffickingdiseases (e.g., cholesterol storage diseases), more preferably,cytotoxic oxysterol accumulation exemplified in NPC disease. In someembodiments, the method is employed to screen neonatal subjects for NPCdisease. The methods of the present disclosure can be used to identifyneonatal subjects that have elevated levels of an oxysterol biomarkerabove a reference value i.e., a value of 24-HC and/or 7-keto and/ortriol concentration above a reference value of each of the oxysterolconcentration from non-NPC afflicted controls, indicative of themetabolic defect in NPC disease. The method for screening a neonatalsubject for NPC disease comprises the steps of: (a) obtaining abiological sample from the neonatal subject; (b) quantifying theconcentration of an oxysterol comprising 24-hydroxycholesterol,7-ketocholesterol, cholesten-3β, 5α, 6β-triol or combinations thereof inthe biological sample; (c) providing a reference value of the oxysterolfrom a non-affected neonatal control population, wherein the referencevalue is the concentration of the oxysterol obtained from the same typeof biological sample as obtained from the subject; and (c) comparing theconcentration of the oxysterol of the neonatal subject to the referencevalue, wherein if the concentration of the oxysterol from the neonatalsubject is higher than the reference value, the neonatal subject isidentified as afflicted with NPC disease.

In some embodiments, the present disclosure also provides methods formonitoring the progression and/or remission of NPC disease. The severityof the NPC disease can be monitored in a subject or patient by measuringthe increase in one or more oxysterol species and the decrease of theoxysterol 24-HC corresponding to the disease. In some embodiments, theprogression and/or remission of NPC disease can be measured bymonitoring the concentration of one or more oxysterols, for example,24-HC, 7-keto and triol.

In some embodiments, the increase in one or more oxysterols including7-keto and triol described herein correlates with NPC disease severity.In a more specific embodiment, the level of 24-HC can be used to monitorthe progression of NPC disease. The progress of NPC disease has beenfound to be inversely correlated with the concentrations of 24-HC afteran initial elevation of 24-HC concentration above a 24-HC referencevalue obtained from a non-NPC afflicted control population.

The present disclosure also provides methods for evaluating the efficacyof treatment by monitoring the concentration of one or more oxysterols,for example, 24-HC, 7-keto and triol during the course of treatment. Insome embodiments, treatment efficacy is generally correlated with adecrease in the concentration of 7-keto and/or triol oxysterol species.Specific method steps can include (a) obtaining a biological sample froma subject at a time (T₀) prior to or while being treated; (b) obtaininga biological sample from the subject at a time (T₁) subsequent to time(T₀); (b) quantifying the concentration of an oxysterol comprising24-hydroxycholesterol, 7-ketocholesterol, cholesten-3β, 5α, 6β-triol orcombinations thereof in the biological samples obtained at (T₀) and(T₁); and (c) comparing the concentration of the oxysterol present inthe biological samples obtained at T₀ and T₁: wherein if theconcentration of the oxysterol 7-ketocholesterol or cholesten-3β, 5α,6β-triol in the biological sample obtained at time (T) is greater thanthe oxysterol concentration of 7-ketocholesterol or cholesten-3β, 5α,6β-triol in the biological sample obtained at time (T₀) or if theconcentration of the oxysterol 24-hydroxycholesterol in the biologicalsample obtained at time (T₁) is less than the oxysterol concentration of24-hydroxycholesterol in the biological sample obtained at time (T₀),then the treatment is not efficacious.

A decrease in the oxysterol concentration, for example 7-keto and/ortriol, obtained from a subject or patient diagnosed with NPC disease ascompared to an earlier measured 7-keto and/or triol oxysterolconcentration indicates that the treatment is effective. Treatmentefficacy can also be determined by measuring the concentration of 24-HCduring the treatment period. In the initial stages of NPC disease thereis an increase of 24-HC over healthy controls, as the disease progressesand/or increases in severity, the concentration of 24-HC in the subjector patient decreases. In a subject or patient diagnosed NPC, if thetreatment provides an increase or stabilization of 24-HC levels in anNPC patient during the course of treatment over an earlier measuredconcentration of 24-HC, the treatment is said to be effective.

In some embodiments, the reference or control level for each oxysterolcan be based on known concentrations of each oxysterol being tested inhealthy and affected populations. The reference or control levels may beset as appropriate for the subject being screened. For example,oxysterol levels in a sample from a neonatal subject can be comparedwith oxysterol levels in a healthy and/or affected neonatal population.In other embodiments, the oxysterol levels identified in a subject canbe compared with a matched unselected, population. In some embodiments,the subject can be compared with a matched population of unaffected(i.e., healthy) subjects and/or a matched population of affectedsubjects. In some embodiments, the subjects can be compared with anage-matched population.

Those skilled in the art will appreciate that some oxysterol levels maybe higher in patients with early onset of NPC disease as compared withlater onset forms of the disease.

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is notintended to limit the present disclosure, application, or uses.

A. Oxysterols: Biomarkers for NPC Disease and Other NeurodegenerativeDisorders.

Without being bound by theory, and without limiting the scope of theinvention, it is presently hypothesized that the presence of elevatedlevels of oxysterols in peripheral and neural cells occurs as a resultof autooxidation or normal enzymatic degradation of cholesterol, as aproduct of oxidative stress, and through dietary intake. With respect to24-HC, it is presently hypothesized that this particular metabolite,also known as cerebrosterol, is produced as a result of24(S)-hydroxylation of cholesterol in the cerebral cortex, hippocampus,dentate gyrus and thalamus.

The production of 24-HC is presently hypothesized to occur as a naturalmechanism for cholesterol release from the brain, and the exchange of24-HC from the brain to the circulation is thought to be a continuousage-dependent flux of about 4 mg/day. Other hydroxylases in the neuronmay also be responsible for the conversion of cholesterol to 7-keto.Evidence presently suggests that the increased levels of the oxysterols24-HC, 7-keto and triol in the plasma and cerebrospinal fluid mightcorrelate with reactive species generation and 24-HC, 7-keto and triolindicative of lipid trafficking defects in neurons and other peripheraltissues, for example, the liver, spleen, kidney and in leukocytes. It istherefore presently hypothesized that elevated oxysterol concentrationsin body fluids (e.g., blood, plasma, serum, cerebrospinal fluid, cellmembrane and the like) and tissues correlates with and is indicative oflipid trafficking defects in neurons, and in particular, indicative ofNPC disease. The oxysterols presently identified as being correlatedwith NPC disease are illustratively shown in FIGS. 1A, 1B and 1C.

B. Identifying and Screening Subjects for NPC Using OxysterolBiomarkers.

In some embodiments of the present disclosure, a method for identifyinga subject for NPC disease is provided comprising quantifying ordetermining the concentration of one or more oxysterols in a biologicalsample obtained from the subject. The concentration of the one or moreoxysterols in the tested biological sample collected from the subjectcan be compared with a reference value. Detection and quantification ofelevated concentration(s) of one or more oxysterol(s) in the biologicalsample as compared to a reference value (which may be a predeterminedvalue) presumptively identifies the subject as affected with NPCdisease.

The reference value represents a threshold value of oxysterolconcentration to which a subject's measured oxysterol levels may becompared. The reference level is selected based on the intended purposeof the test or assay being performed on the subject. For example, areference level may be determined for purposes of identifying subjectsaffected by NPC while the same or different reference level may be usedfor monitoring the progression or severity of disease in a subject, orfor screening a particular population of subjects for the disease, forexample, neonatal subjects.

The selection of an appropriate reference level for a given test orassay is within the level of skill in the art. The choice of thereference value is not absolute. For example, a relatively low value mayadvantageously be used to reduce the incidence of false negatives, butmay also increase the likelihood of false positives. Accordingly, as forother screening techniques, the reference value may be based on a numberof factors, including but not limited to cost, the benefit of earlydiagnosis and treatment, the invasiveness of follow-up diagnosticmethods for individuals that have false positive results, the intendedpurpose of the test or assay, the limits of accurate detection ofoxysterol levels in the particular sample type, the prevalence andaverage or mean oxysterol levels of the relevant population, the desiredlevel of statistical significance of the results, the level ofquantification desired in terms of disease risk, presence, progression,or severity, and other factors that are routinely considered indesigning screening assays.

As a non-limiting example of the selection of a reference value,subjects that have one or more of the oxysterol biomarkers selected from24-1HC, 7-keto and triol having values above about the 70th percentile,75^(th) percentile, 80th percentile, 90th percentile, 95th percentile,96th percentile, 97th percentile, 98th percentile, 99th percentile, orhigher, as compared with an appropriate control population may bepresumptively identified as affected with NPC disease. In someembodiments, the control population can be a matched control populationwherein the control population is matched to the subject by genderand/or age. Alternatively, subjects having more than about 2, 3, 4, 5,8, 10 or 20 fold higher oxysterol biomarker concentration than theaverage (alternatively, mean or median) value for an appropriateunaffected population as measured under similar or identical conditionsmay be presumptively identified as affected with NPC disease. However,in essentially most cases, the diagnosis or identification of NPCcorresponds to an oxysterol concentration that is above the referencevalue, even if the identified concentration of oxysterol in thebiological sample of the NPC identified subject is not at least 2 foldhigher than the reference value.

An appropriate reference value may be determined in a variety of ways.In some embodiments, the reference value can be a predetermined value,based for example, on prior tests and assays on healthy or affectedsubjects ranging from six individuals to greater than a thousand. Inother embodiments, the reference value can be determined during thecourse of the assay. For example, samples from known unaffected and/oraffected subjects may be run concurrently with the test samples and areference value determined therefrom. In yet other embodiments, testsamples from a mixed population can be analyzed, and the reference valuecan be determined based on the distribution of the results usingstatistical methods known in the art.

In general, the methods for identifying, screening or monitoring theprogression and/or treatment efficacy disclosed herein haveexperimental, veterinary and medical applications. Accordingly, subjectsmay be humans, primates, simians, canines, felines, equines, bovines,ovines, caprines, porcines, lagomorphs, rodents, avians, and the like.Preferably, however, subjects according to the present disclosure willbe human subjects, e.g., neonatal (i.e., from the time of birth to aboutone week post-natal), infant, juvenile, adolescent or adult subjects. Insome embodiments, neonatal subjects can be used for screening purposes.As used herein, “neonatal” subjects can include premature infants, asthat term is used in the art.

The subjects may be part of a general population, e.g., for abroad-based screening assay. Alternatively, the subject may be one thathas an enlarged liver or spleen, or is suspected of having a lysosomalcholesterol storage or trafficking disorder characterized by one or moreclinical symptoms. The subject may also be experimentally determined tobe accumulating one or more oxysterols (e.g., the subject has clinicalsymptoms) as described above (e.g., elevated expression of one or moreoxysterols in one of blood, plasma, serum, cerebrospinal fluid and cellmembranes (for example, red blood cell membranes). In other embodiments,subjects have already been diagnosed as having NPC disease, for example,by detection of accumulation of one or more of 24-HC, 7-keto and trial,and the clinical condition of the patient and/or the efficacy of thetreatment may be monitored. Methods for extracting or obtaining abiological sample of each of the subjects described above are well knownin the art of medical and veterinary science, for example, phlebotomy,heel stick of an neonatal subject or infant or a tissue biopsy.

As used herein, the “biological sample” can include any body fluid ortissue from the subject, for example, blood, plasma, serum, cells,biopsy samples, or other tissue (including in vitro cultured cells) inwhich oxysterol accumulation may be detected and quantified. Thesuitability of a particular body fluid or tissue for use in the methodsdescribed herein may be determined by testing a sample of the fluid ortissue obtained from a subject suspected of having, diagnosed with orscreened positively for a lysosomal cholesterol storage or traffickingdisease (e.g., cholesterol storage diseases) including cytotoxicoxysterol accumulation (NPC).

The biological sample may be obtained by surgical methods or biopsy.More preferably, the biological sample may be obtained by relativelynon-invasive methods, which are less traumatic to the subject, and moresuitable for a broad-based screening assay. In some embodiments, thebiological sample is a body fluid sample. Illustrative examples caninclude body fluid samples, including, but not limited to, plasma, sera,blood (including cord blood), sputum, amniotic fluid, and the like.Blood, plasma, sera and cellular membrane samples (for example red bloodcell membranes) are more preferred.

Alternatively, the biological sample is a cell or tissue sample,including cultured cells (e.g., fibroblasts) or tissues, and conditionedmedium or effusions collected from cells or tissues. Exemplary cells ortissues can include, hematopoietic cells (including red blood cells,leucocytes, lymphocytes and the like), neural cells (including neurons,microglial cells and astrocytes), muscle (including skeletal, smooth,cardiac and diaphragm), liver, kidney, lung, skin, foreskin, umbilicalcells or tissue, and the like.

In some embodiments, the biological sample can be provided on a solidmedium, e.g., a filter paper, swab, cotton, and the like. In someembodiments, the biological sample is a dried blood or plasma samplefrom a neonatal subject, e.g., dried blood or plasma spots on neonatalscreening cards (i.e., “Guthrie” cards).

Subjects may be presumptively identified as affected with NPC disease bythe identifying and screening methods described herein. In someembodiments, additional, second-tier diagnostic testing can be carriedout to confirm the diagnosis in these subjects. Typically, suchsecond-tier methodologies (e.g., restriction fragment lengthpolymorphism (“RFLP”), polymerase chain reaction (“PCR”) and othergenetic tests on tissue biopsies) are more costly, time-consuming andinvasive than the screening methods disclosed herein. For example,subjects having one or more oxysterol levels above a reference value maybe presumptively identified as affected with NPC disease, and selectedfor additional diagnostic testing to confirm this diagnosis, determinethe severity of the disease, assess whether the subject is affected withanother neurodegenerative disorder or lysosomal cholesterol storagedisorder, or is a healthy subject giving a false positive result in thescreening assay. Other tests for confirming the identification of asubject as having NPC disease using the methods of the presentdisclosure can include one of three commonly used tests. First, a skinbiopsy can be obtained from the presumptively identified NPC diseaseafflicted subject. The skin fibroblasts are cultured and then stainedwith filipin to detect lysosomal accumulation of unesterifiedcholesterol. This assay is qualitative but not quantitative. Second, anassay which determines the rate of cholesterol esterification can bemeasured in the cultures skin fibroblasts. Low rates of esterification,as compared to reference cell lines, are indicative of NPC disease.Third, the gold standard assay to confirm NPC disease currently usedincludes sequence analysis of the NPC1 and NPC2 genes of the. Thismethod detects >95% of clinically suspected NPC cases. Because thegenomic regions are not sequenced, a small percentage of NPC cases withnovel intronic mutations may escape detection.

C. Monitoring Course, Severity, or Clinical Status of NPC UsingOxysterol Biomarkers.

In another embodiment, the invention provides methods for monitoring theclinical course of a subject that has already been positively diagnosedand/or is being simultaneously diagnosed as affected with a disordercharacterized by the accumulation of an oxysterol, (for example at leastone of 24-HC, 7-keto and triol), as this term is described above. Thepresent disclosure provides that elevated oxysterol concentrations inbiological samples (in particular, plasma, blood and sera) from affectedsubjects correlate with the clinical state of the affected subject.Moreover, oxysterol concentrations may be elevated prior to theworsening of the disease in the affected subject, and thus may be usedas an early indicator of regression. Thus, oxysterol levels may be usedas an index of treatment efficacy and the clinical condition of thepatient.

In some embodiments, the level of 24-hydroxtcholesterol inverselycorrelates with disease severity after initial elevation. The24-hydroxycholesterol level can be used to measure treatment efficacyand the clinical condition of the patient (See FIG. 3). Accordingly, thepresent disclosure further encompasses methods of monitoring theclinical status of a subject with a disorder characterized by theaccumulation of oxysterols 7-keto and triol or the reduction in 24-HC inthe subject. In some embodiments, the subject has already been diagnosedwith NPC disease.

The clinical condition of the subject may be monitored to determine theefficacy of a treatment regime, e.g., enzyme replacement therapy, genetherapy, pharmaceutical intervention (for example treatments with one ormore of: statins or other cholesterol synthesis inhibitors,cycloheximide, liver X receptor (LXR) agonists, orphan nuclear receptorPXR ligands, neurosteroids, N-butyldeoxynojirimycin (NB-DNJ)(miglustat), curcumin, chemical chaperones, antibiotics, salsalate,salicylic acid, RXR ligands, sphingolipid synthesis inhibitors(myriocin), KCl, EGTA, calcium channel inhibitors, nifedipine,verapamil, antioxidants, vitamin E, vitamin C, aurinticarboxylic acid,flavanoids, cyclodextrins, estrogens, propyl gallate, glutathione,caspase inhibitors, MAP kinase inhibitors, peroxisomeproliferator-activated receptor (PPARs) ligands, 15d-PGJ2, WY 14643,indomethacin, glucocorticoids, dexamethasone, hydrocortisone, PI-3kinase inhibitors, NMDA open channel blockers) and/or dietary therapy.For example, if the level of the oxysterol biomarker suggests that thecurrent therapeutic regime is not effective, or not efficacious, it maybe determined to initiate an altered course of treatment. Alternatively,the condition of the subject may be monitored to determine whether tocommence or re-initiate treatment of the subject.

In some embodiments, the present disclosure provides a method todetermine the efficacy of treatment for Niemann-Pick C disease in asubject. The method includes: (a) obtaining a biological sample from asubject at a time (T₀) concurrent with or prior to the commencement oftreatment; (b) obtaining a biological sample from the subject at a time(T₁) subsequent to the commencement of treatment; (c) quantifying theconcentration of an oxysterol comprising 24-hydroxycholesterol,7-ketocholesterol, cholesten-3β, 5α, 6β-triol or combinations thereof inthe biological samples obtained at (T₀) and (T₁); and (d) comparing theconcentration of the oxysterol present in the biological samplesobtained at T₀ and T₁. If the concentration of the oxysterol7-ketocholesterol or cholesten-3β, 5α, 6β-triol in the biological sampleobtained at time (T₁) is greater than the oxysterol concentration of7-ketocholesterol or cholesten-3β, 5α, 6β-triol in the biological sampleobtained at time (T₀) or if the concentration of the oxysterol24-hydroxycholesterol in the biological sample obtained at time (T₁) isless than the oxysterol concentration of 24-hydroxycholesterol in thebiological sample obtained at time (T₀), then the treatment is notefficacious.

The time lapsing between (T₀) and (T₁) can be determined by those ofordinary skill in the art, in particular, the subject's medicalprofessional. While not wishing to be bound by theory, it is believedthat altered expression of oxysterol biomarkers 24-HC, 7-keto and triolcan be detected and measured as soon as 1 week. Therefore, the methodfor determining the efficacy of a treatment for Niemann-Pick C diseasein a subject can include taking a biological sample at time 0 (T₀)before, or concurrently with the commencement of treatment for NPCdisease. Then the efficacy of the treatment can be determined byquantifying the concentration of one or more oxysterols using themethods described herein at a time (T₁) which may be at least 1 week, orat least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least8 weeks or at least 3 months, or at least 6 months after thecommencement of treatment.

Preferred methods of quantifying or determining 24-HC, 7-keto and trioland other oxysterols in biological samples using TMS are described inmore detail hereinbelow.

D. Methods for Quantifying Oxysterol Concentration in Biological Samples

In some embodiments, the methods for quantifying oxysterol concentrationon a biological sample can be simple, rapid, accurate, relativelynon-invasive (e.g., non-surgical), sensitive, and preferably minimizeinterfering signals from molecules other than the oxysterols 24-HC,7-keto and triol. The method for quantifying the concentration of theoxysterol can include: (a) adding a known amount of an oxysterolinternal standard to a biological sample; (b extracting the oxysterolsfrom the biological sample: and (c) quantifying the extracted oxysterolsusing a chromatography procedure.

As used herein, a chromatography procedure or combination ofchromatography procedures can be used to quantify the oxysterolconcentration in the collected biological sample. Methods for isolatingsterols, including cholesterol and oxidized cholesterol are known in theart. In some embodiments, the quantification step of the method ofidentifying a subject with NPC disease can include: determining therelative concentration of the oxysterol and internal standard in thebiological sample by correlating the area under the curve obtained forthe known amount of oxysterol internal standard with the area under thecurve obtained for the one or more oxysterols. Such determination can beaccomplished when the isolated and/or derivatized oxysterols are passedthrough a chromatography procedure operable to derive the relativeabundance of each oxysterol being identified relative to the knownamount of oxysterol internal standard. The relative quantities of eachof the oxysterols and oxysterol internal standard separated and/orisolated during the chromatography procedure can be routinely determinedusing computers and other processing devices that can be integrated withthe detection means used in the chromatography equipment, for example,infra red, visible light diffraction, UV detection, mass spectroscopy,mass ionization and other methods used to detect lipids, sterols and/oroxysterols passing through a chromatography column or component.

In some embodiments, it is preferable that the oxysterol quantificationmethodology be compatible with existing screening assays and isadaptable to automation and high throughput screening. Methods useful todetermine the concentration of one or more oxysterols in a biologicalsample indicative of NPC and other lysosomal cholesterol storage andtrafficking diseases (e.g., cholesterol storage diseases), may becarried out using any suitable methodology or combination ofmethodologies that detects the presence or absence of oxysterols, andpreferably, methodologies which determine the concentration of theoxysterol. Illustrative methods include, but are not limited to,chromatographic methods (e.g., high performance liquid chromatography(“HPLC”)), thin layer chromatography (“TLC”), liquid chromatography-massspectrometry (“LC-MS”): gas chromatography-mass spectrometry (“GC-MS”),time-of-flight mass spectrometry (“TOF-MS”), tandem mass spectrometry(“TMS”), matrix assisted laser desorption ionization—mass spectrometry(“MALDI-MS”), electrospray ionization—tandem mass spectrometry(“EIS-TMS”) and combinations of these mass spectrometry techniques withor without sterol derivatization. Schroepfer Jr., G. J, “Oxysterols:Modulators of Cholesterol Metabolism and Other Processes”, (2000).Physiol. Rev. 80(1): 362-521, provides additional assays and methodswhich may be useful in oxysterol quantification and identification. Insome embodiments, HPLC, GC/MS, TOF-MS, and EIS-TMS can be used todetermine the concentration of oxysterols in a biological sample.Further examples of quantification of oxysterol concentration from invivo biological samples are described herein in the Examples section.

Jiang, X., et al. “Characterization of oxysterols by electrosprayionization tandem mass spectrometry after one-step derivatization withdimethylglycine” Rapid Commun. Mass Spectrom., (2007); 21:141-152,describes high throughput screening methods for the detectionidentification and quantification of oxysterols using ESI-TMS. Such highthroughput screening methods are useful in some embodiments of thepresent disclosure.

In some embodiments, the present disclosure provides methods that can becompletely manual, alternatively and preferably, they are partially orcompletely automated. Screening programs to determine the concentrationof oxysterols in a large number of biological samples, for example, aneonatal screening regime will generally be at least partially automatedto facilitate high throughput of samples. The data can be captured andanalyzed using an automated system. Other illustrative examples of highthroughput methods can include arrays or micro-arrays of spottedbiological samples (e.g., blood, plasma, serum, red blood cells and thelike) on substrates which can be analyzed simultaneously. Such arrays ormicroarrays can contain greater than about 10, 50, 100, 200, 300, 500,800, 1000, 2000, 5000 samples or more.

In biological samples in which the concentration of the oxysterol is lowrelative to the limits of detection of the technique, it is preferred touse a derivatization step prior to the step of detecting (alternatively,quantifying) oxidized cholesterol in the biological sample. In someembodiments, the derivatization step can also be performed after theoxysterols have been isolated or concentrated to yield higher quantitiesof oxysterols for quantification. The derivatization of oxysterols inthe biological sample facilitates proper identification using thechromatography and mass spectrometry methods such as HPLC, LC-MS, GC-MS,TOF-MS, and EIS-TMS and may also be used to separate the oxysterolanalyte from contaminants or interfering substances. Jiang, et al. supraprovides additional protocols for the derivatization of oxysterolspecies in mixtures using ESI-TMS which may be useful in the presentdisclosure.

Referring to FIG. 2, the table below each mass spectra illustrates therelative concentration of each oxysterol which can be determined bycalculating the area under the curve of each tested oxysterol in controland NPC affected representative samples relative to known amounts of anoxysterol internal standard, for example, D5-27-hydroxycholesterol. Suchcalculations can be automated and processed by the chromatographicsoftware used. Relative concentrations of oxysterols found in variousbiological samples such as plasma (FIGS. 3A & 3B) and cerebrospinalfluid (FIG. 4) from NPC subjects and healthy control subjects using themethods described herein, illustrate the selectivity and specificity ofthe oxysterols 24-HC, 7-keto and triol as exquisite biomarkers for NPCdisease.

E. Neonatal Screening.

The methods disclosed herein may be advantageously employed as part of aneonatal screening program to identify affected neonatal subjects, forexample, in the neonatal intensive care unit so as to permit earlymedical intervention. Many neonatal screening programs rely on a uniquemethod of specimen collection, in which blood from a heel prick isabsorbed onto a neonatal screening card (e.g., a cotton-fiber filterpaper). Several neonatal diseases are screened in these subjects duringtheir first weeks of life, including phenylalanine hydroxylasedeficiency, which causes phenylketonuria (“PKU”), and branched-chainketoacid dehydrogenase deficiency, which causes maple syrup urinedisease (“MSUD”). Such screening programs are effective and cause no orlittle discomfort to these delicate patients.

The neonatal screening methods disclosed herein, including highthroughput screening assays may further advantageously be performedconcurrently or in parallel (i.e., from the same sample but notnecessarily in the same assay) with other neonatal screening assays, forexample, on neonatal blood samples or dried blood spots on neonatalscreening cards. In some embodiments, a neonatal screening program canbe based on quantifying or measuring oxysterols from other biologicalsamples, as described above. For example, 24-HC. 7-keto and triol can bemeasured in blood (e.g., cord blood), plasma, serum, or in the membraneof the neonatal blood cells, for example red blood cells. In someembodiments, one blood draw from a neonatal subject can providesufficient biological sample to validate the quantity of one or moreoxysterols present in different biological samples, for example, plasmaand red blood cells using the same or different testing methods.

Accordingly, in some embodiments of the present disclosure, a method forscreening a neonatal subject for a disorder characterized by theaccumulation of one or more oxysterols (as described above), comprisingthe step of quantifying or determining the concentration of oxysterol ina biological sample taken from the neonatal subject, wherein thedetection of the oxysterol in the biological sample a concentration thatis greater than a reference value of the same oxysterol identifies theneonatal subject as affected with the disorder.

In some embodiments, the method of screening a neonatal subject for NPCdisease comprises the step of quantifying or determining theconcentration of oxysterol in a blood sample taken from the neonatalsubject, wherein the detection of the oxysterol in the biological sampleat a concentration that is greater than a reference value identifies theneonatal subject as affected with NPC disease.

The method can include the steps: (a) obtaining a biological sample fromthe neonatal subject; (b) quantifying the concentration of an oxysterolcomprising 24-hydroxycholesterol, 7-ketocholesterol, cholesten-3β, 5α,6β-triol or combinations thereof in the biological sample; (c) providinga reference value of the oxysterol from a non-affected neonatal controlpopulation, wherein the reference value is the concentration of theoxysterol obtained from the same type of biological sample as obtainedfrom the subject; and (d) comparing the concentration of the oxysterolof the neonatal subject to the reference value, wherein if theconcentration of the oxysterol from the neonatal subject is higher thanthe reference value, the neonatal subject is identified as afflictedwith Niemann-Pick C disease.

In still further embodiments, methods as applied to diagnosing,monitoring of disease severity and determining the efficacy of treatmentdescribed above can also be applied to neonatal subjects.

In still a further embodiment, described in more detail below, methodsfor quantifying an oxysterol concentration is a biological sampleinvolving tandem mass spectrometry are utilized as part of a neonatalscreening program for NPC disease using oxysterols 24-HC, 7-keto andtriol as biomarkers for the presence of the disease.

As described above, it is preferred that methods of neonatal screeningbe at least partially automated. For example, once a sample is loadedonto an HPLC column or into a mass spectrometer, it is preferred thatthe data be captured and analyzed using an automated system.

F. Methods for Quantifying an Oxysterol in a Biological Sample UsingTandem Mass Spectrometry (TMS)

In some embodiments, the present disclosure further provides a methodfor quantifying or determining the concentration of oxysterols in abiological sample by ESI-TMS. In some embodiments, the oxysterolspresent in the biological sample (blood, plasma, serum or cell membrane)will need to be extracted and isolated from other substances; includingsubstances that may interfere with the detection and/or quantificationof oxysterol biomarkers. Methods for extracting oxysterols from complexmixtures are well known in the art. Conventionally, extraction of lipidsfrom complex biological samples commences with a two-phase extractionwith chloroform and methanol. In some embodiments, the organic phase ofthe two-phase extraction containing the oxysterols may be furtherpurified using chromatography steps such as HPLC or passage throughaminopropyl and silica columns to isolate a neutral sterol fractionprior to derivatization. Derivatization of the oxysterols in the samplemay be performed by any method known in the art, includingderivatization using N,N-dimethylglycine, Girard P reagent andPyridine:Hexamethyldisilazane:Trimethylchlorosilane in ratios of 5:2:1.In some embodiments, oxysterols can be saponified under mild conditionsto avoid artificial oxysterol generation and derivatized withbis-(trimethyl-silyl)-trifluoroacetamide (BSTFA) and pyridine intotrimethylsilyl ethers.

In some embodiments, matrix assisted laser desorption ionization—time offlight mass spectrometry (“MALDI-TOF MS”), GC-MS, LC-MS or ESI-TMS canbe used to carry out the inventive methods described herein. Jiang, et.al. supra provides additional protocols for the preparation,derivatization and identification of oxysterol species in mixtures usingESI-TMS which may be useful in the present disclosure.

Any suitable MS methodology known in the art may be employed, including,but not limited to GC-MS, LC-MS, Liquid Chromatography/AtmosphericPressure Chemical Ionization Mass Spectrometric (“LC/APCI-MS”), ESI-MS,MALDI-MS and MALDI-TOF MS. Electrospray ionization, ion traps and ioncyclotron resonance equipped mass spectrometers can also be employed.

Further disclosed herein is an ESI-TMS protocol for screening for NPCdisease using one or more oxysterols as biomarkers to identify a subjectas haring NPC or to be used as a screening method, for example, aneonatal screening method before the onset of clinical symptoms.

For the selective detection of compounds of a similar structural type,either a precursor ion scan function to identify the molecular speciesthat fragment to a common product ion, or a constant neutral loss scanfunction to identify ions that lose a common fragment, or a multiplereaction monitoring where selected precursor and product ions only aredetected can be employed. Addition of appropriate oxysterol internalstandards, such as stable analogs, to the biological matrix beforework-up and analysis facilitates accurate quantification of the targetoxysterols.

An oxysterol internal standard is generally added to the sample prior tomanipulations, so that the standard is subjected to the same conditionsas the analyte. Any suitable internal standard may be used. Oxidizedcholesterol homologs in which one of the carbons, preferably at position27, is hydroxylated (for example, D₅-27-hydroxycholesterol) is suitableand preferred. The internal standard can be added to the sample in aknown quantity. The ratio of signals produced by 24-HC, 7-keto, trioland other oxysterols that are elevated in NPC subjects and the internalstandard will allow the starting quantity of the oxysterol biomarkers inthe sample to be determined by use of a calibration curve. Thecalibration curve is a plot of the signal ratio (oxysterol biomarkers ofNPC to internal standard) against different known concentrations of24-HC, 7-keto, triol standards, using the same fixed quantity ofinternal standard. In some embodiments, peak identification can beconfirmed by relative retention time and mass spectral comparison withone or more authenticated oxysterol standards, as well as with the HP MSChemstation NBS Mass Spectral Data Library of compounds.

In some embodiments of the present disclosure, a method for quantifyingor determining oxysterol biomarkers in a biological sample comprises:(1) collecting a biological sample; (2) adding a known quantity of asuitable oxysterol standard to the sample; (3) extracting the oxysterolsfrom the sample using a two-phase extraction medium; (4) purifying theoxysterol in the sample by normal phase chromatography, followed byelution from the column with a suitable solvent; (5) derivatization ofthe oxysterol and (6) quantification of the oxysterol biomarker usingMS. Preferably, a D₅-27-hydroxycholesterol standard is used as aninternal standard for the MS analysis.

The foregoing methodology can be employed in preferred embodiments ofthe inventive screening and testing methodologies described above. Asfurther described above, it is preferred that the methods be partiallyor completely automated.

TMS based methodologies are particularly suitable for quantifying ordetermining oxysterols 24-HC, 7-keto and triol as biomarkers for NPCdisease in dried blood spots from neonatal screening cards. According tothis embodiment, the method above further comprises a step of extractingthe lipid component from the dried blood spot using a suitable solvent(e.g., an organic solvent or aqueous/organic mixture).

Thus, in some embodiments, the present disclosure provides a method ofscreening a neonatal subject for NPC disease, comprising: (1) providinga blood sample, comprising a dried blood spot on a solid absorbentsubstrate (e.g., a filter paper); (2) adding a known quantity of asuitable oxysterol standard to the sample; (3) extracting the oxysterolsfrom the sample using a two-phase extraction medium; (4) purifying theoxysterol in the sample by normal phase chromatography, followed byelution from the column with a suitable solvent; (5) derivatization ofthe oxysterol and (6) quantification of the oxysterol biomarker usingMS. Preferably, a D₅-27-hydroxycholesterol standard is used as aninternal standard for the MS analysis; and (7) presumptively identifyingthose subjects as affected with NPC disease based on oxysterolconcentrations in the biological sample that are greater than areference value (as described above).

EXAMPLES Example 1 Isolation of Oxysterols from NPC Patient Samples

200 pmol of d₅-27-hydroxycholesterol (27-HC), 50 μg butylatedhydroxytoluene (BHT) and a magnetic flea bar are added to a 10 mLcentrifuge tube. The tube is placed under a gentle stream of argon. 250μL of plasma from a subject and 2.5 mL 0.35 M KOH is added to the tube.The tube is sealed tightly with a Teflon-lined screw cap, vortexed, andallowed to incubate for 2 hr at room temperature while stirring. Afterincubation, the flea bar is removed and the tube is centrifuged at 3000rpm for 5 min to pellet the protein. The liquid phase of plasma mixtureis removed and added to a 10 mL centrifuge tube placed under argonstream. The tube is then sealed tightly. The pH of liquid phase islowered to approximately 7.0-8.0 using a 1:10 dilution of 85% H₃PO₄.While vortexing the centrifuge tube, 200 μL of H₃PO₄ is added dropwiseto the plasma mixture until desired pH is achieved. 1.54 mL of 150 mMNaCl and 4.5 ml. CHCl₃ is added to the plasma mixture. The tube issealed with a Teflon screw cap, vortexed, and centrifuged at 3400 rpmfor 5 min. The lower organic phase is extracted, transferred to a new 10mL centrifuge tube, and placed under argon stream. 2.0 mL of CHCl₃ isadded to the remaining aqueous phase of the first extraction, vortexed,and then centrifuged. The organic phases are collected from thecentrifuge tubes and transferred to clean tubes under argon. Theextraction steps above are then repeated. The pooled organic phases areevaporated under argon in a clean tube and sealed tightly with aTeflon-lined cap. The samples are then ready for oxysterol purification.

Example 2 Oxysterol Purification Using Aminopropyl and Silica Columns

The extracted lipid samples are resuspended in 250-300 μL CHCl₃ and thenvortexed. One or more aminopropyl columns (Sep-Pak Vac RC 500 mg NH₂Cartridges, part no. WAT054515) are primed with 4 mL hexane and areloaded with the lipid sample in CHCl₃. The sample drips into the columnusing gravity. The neutral fraction is eluted with 4 mL CHCl₃/isopropyl(2:1 v/v) and then dried under argon. The neutral fraction isresuspended in 1 mL toluene and the fraction is added to a silica column(Isolute 100 mg SI 100 mL XL cartridges, part no. 460-0010-G) primedwith 4 mL hexane. The neutral fraction volume is pulled through thesilica column (without drying out) using vacuum. The column is washedwith 8 mL of 0.5% isopropyl in hexane, followed by elution of theneutral fraction with 2 mL of 30% isopropyl in hexane. The elutedfraction is collected and dried under argon. After silica columnpurification, the sample is resuspended in 250 μL of acetonitrile andtransferred to a low retention/silanized microcentrifuge tube. The tubecontaining the eluted fraction is again washed with 250 μL acetonitrileand the resuspension added to a second microfuge tube. The elutedfraction containing the oxysterol component is then dried and sealedunder argon.

Example 3 Derivitization of Oxysterols from Plasma Samples

To derivatize the oxysterols, a solution of 5:2:1 (v:v:v) of Pyridine;Hexamethyldisilazane:Trimethylchlorosilane is mixed in a centrifugetube, vortexed, and the solution is centrifuged at 3400 rpm for 2 min.100 μL of the derivitization solution is added to each of the samplesprepared above containing the oxysterol component in eppendorf tubes.The eppendorf tubes are then sealed tightly, vortexed, and thederivatization solution and samples are incubated at 60° C. for 30 min.The reaction product containing the derivatized oxysterols is then drieddown under a stream of argon. The derivatized oxysterols are resuspendedin 100 μL heptane and the resuspension is then vortexed, centrifuged andtransferred to an autosampler vial.

Example 4 Analysis of Oxysterol Profile by Gas Chromatography-MassSpectrometry

The samples containing the derivatized oxysterols are run on an AgilentTechnologies 5975B inert XL MSD with an Agilent Technologies 6890NNetwork GC System equipped with an Agilent Technologies 7683 Autosamplerand an Agilent Technologies 7683B injector. A J&W Scientific 25 m modelDB-1 column with an internal diameter of 0.2 mm and film thickness of0.33 μm is used to separate the oxysterol species. 2.0 μL of sample isinjected into the inlet, which is kept at 250° C. with a pressure of28.98 psi. A split ratio of 10:1 is used, with a split flow of 10.6mL/min and a total flow of 15 mL/min within the inlet. The oven startsat 180° C. prior to ramping up to 250° C. at a rate of 20° C./minfollowed by an additional ramp up to 300° C. at a rate of 5° C./minwhere it stays for 15 min, allowing for an overall run time of 28.5 min.Initial flow of gasses through the column is set at 1.0 mL/min allowingfor a nominal initial pressure of 28.04 psi and average velocity of 42cm/sec. The MSD transfer line is kept at 280° C. throughout the samplerun. The MS quadrupole is set to 150° C. and the MS source is set to230° C. The MSD is set to monitor for 461.4, 544.4, 472.4, 456.4, and413.4 ions at dwell times of 61 ins. FIG. 2 shows a gaschromatogram/mass spectrum (GC/MS) of oxysterols in plasma samplesobtained from confirmed NPC patients and control non-NPC afflictedsubjects.

Results

Thirty-three plasma samples were obtained from subjects with NPC diseaseenrolled in the natural history study at the National Institutes ofHealth. The plasma samples were collected in “purple-top” tubescontaining K₃-EDTA and BHT. The tubes were immediately centrifuged andthe plasma was isolated, stored in 1 ml aliquots and frozen at −80° C.The plasma samples were thawed immediately prior to analysis and thesamples were processed as described above. The NPC plasma samples wereanalyzed for levels of d5-27-HC (internal standard), 27-HC, 24-HC(24-hydroxycholesterol), 7-ketocholesterol (7-keto),Cholesten-3β,5α,6β-triol (triol), and 7α,27-HC (See FIG. 2.). Theresults are presented in table 1 below:

TABLE 1 Oxysterol quantification in control and NPC confirmed subjectsfrom plasma samples using GC-MS. Oxysterol NPC (ng/mL) Control (ng/mL) pvalue 7□, 27 9.79 9.95 0.875 7-keto 455.14 106.06 2.98E−12 27-HC 93.20117.41 0.132 24-HC 69.49 33.08 2.63E−09 triol 174.41 8.88 5.46E−17

A two-sample t-test assuming equal variance was used to test forsignificance between NPC patients and normal controls for 24-HC,7α,27-HC, 27-HC, and 7-keto. A two-sample t-test assuming unequalvariance was used for triol. Three of the oxysterols (24-HC, 7-keto andtriol) were significantly elevated in NPC subjects, as compared tonormal controls, and thus represent plasma biomarkers for diagnosis ofNPC disease.

The relationship between the oxysterol biomarkers and disease severityin the NPC subjects was also examined. Among the selected oxysterols,the concentration of 24-HC revealed a statistically significant inversecorrelation between plasma levels of 24-HC and clinical disease severity(see FIG. 5). The Pearson correlation coefficient obtained was 0.67,with an R² value of 0.45 and a p value=0.0022. The concentration of24-HC in a biological sample may be a useful biomarker for following NPCsubjects longitudinally, and may provide a quantitative biomarker tofollow efficacy of therapeutics interventions in slowing the progressionof neurodegeneration in NPC subjects.

The stability of the oxysterols triol and 7-keto prepared in accordancewith the methods of the present disclosure are shown in FIGS. 7A and 7B.Patient's having confirmed NPC disease were used to obtain plasmabiological samples. The plasma biological samples were processed asdescribed in Examples 1-4 except that some of the samples were nottreated with butylated hydroxytoluene (BHT, a known antioxidant thatprevents further oxidation of cholesterol), and either treated at roomtemperature or at 4° C. with and without BHT. The results show that themethods of the present disclosure can be performed at 4° C. or at roomtemperature and with and without BHT without adversely affecting theability to isolate and quantify the presence of the oxysterols triol and7-keto in the methods steps outlined in Example 1-4.

In addition to testing the stability of the oxysterols when isolated anddetermined using the methods of the present disclosure, the time ofbiological sampling was also measured to determine whether there is adifference in the in vivo production by the subject of the oxysterolsbeing identified. As shown in FIGS. 7A and 7B, there is no statisticallybeneficial or detrimental time to take a biological sample from thesubject to be identified or screened for NPC disease. The data appearsto indicate that the production of oxysterols triol and 7-keto is fairlyconstant throughout a 24 hour day.

Example 5 Specificity of the Oxysterols 7-Keto and Triol to Identify NPCDisease from Other Lysosomal Storage and Trafficking Diseases

Oxysterols 7-keto and triol from a variety of human subjects having alysosomal storage or trafficking disease including those with known CNSinvolvement: infantile neuronal ceroid lipofuscinosis (INCL), GM1gangliosidosis (GM-1), GM-2 gangliosidosis (GM-2) (Tay-Sachs Disease),Gaucher's disease (GD) and hepatosplenomegaly (HSM). As shown in FIGS.6A and 63, the combination of elevated 7-keto and triol levels was ableto differentiate NPC subjects from subjects with other LSDs. Theconcentration of the oxysterol 7-keto and triol are expressed asnanograms per milliliter (ng/mL) of plasma. The results depicted inFIGS. 8A and 8B for the oxysterols 7-keto and triol respectively,demonstrate that for identifying and screening purposes, (e.g. in humansubjects), the oxysterols 7-keto and triol can be used as selectivebiomarkers to identify and screen NPC subjects from other subjectshaving a lysosomal storage or trafficking disease.

Example 6 Determination Of Oxysterols In NPC and Wild-Type Mice

The present methods for quantifying the presence of oxysterols from NPCafflicted subjects, including NPC1−/− mice and wild-type non-afflictedNPC mice were performed to determine the expression of variousoxysterols in these population. Measurements of various plasmaoxysterols in the NPC1-deficient and wild type mice from ages 4-10 weekswere performed. Note that for these oxysterols, the divergence isgenerally greatest after 7 weeks when the mice are most symptomatic.Methods for isolating and quantifying the levels of oxysterols in thewild-type and NPC1 null mice are essentially described herein.

As shown in FIG. 9, significant elevations in plasma25-hydroxycholesterol (25-HC) and cholesten-3β,5α,6β-triol (triol) inNPC1 mice as compared to wild-type mice were observed at all timepoints. Even more striking was the separation in the plasma oxysterollevels (4β-hydroxycholesterol (4β-HC), 7α-hydroxycholesterol (7α-HC),7β-hydroxycholesterol (7β-HC), and 7-keto), between wild-type and NPC1mice beginning at 7-8 weeks of age, when the NPC1 mice begin losingweight and are overtly symptomatic. The elevated plasma oxysterols inthe NPC1 null mice were corroborated by increased oxysterol levels incerebellar and liver tissues. Remarkably, elevated triol levels incerebellar tissue were detected as early as postnatal day 8 in the NPC1mice, when the mice are asymptomatic yet display neuronal cholesterolstorage. Taken together, these findings indicate that plasma levels ofnon-enzymatically generated oxysterols, including 25-HC, triol, 4β-HC,7α-HC, 7β-HC, and 7-keto can be used to distinguish between wild-typeand NPC1 disease, and may vary with disease progression. Theseoxysterols can be used as biomarkers for the identification andmonitoring of NPC disease and its progression.

The foregoing description of the embodiments has been provided forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention. Individual elements or features ofa particular embodiment are generally not limited to that particularembodiment, but, where applicable, are interchangeable and can be usedin a selected embodiment, even if not specifically shown or described.The same may also be varied in many ways. Such variations are not to beregarded as a departure from the invention, and all such modificationsare intended to be included within the scope of the invention.

What is claimed is:
 1. A method for determining efficacy of a therapy ina subject afflicted with a disorder involving the accumulation of one ormore oxysterols, comprising: (a) providing at least one first body fluidsample from the subject prior to administering a therapy to the subject;(b) administering the therapy to the subject; (c) obtaining at least onesecond body fluid sample from the subject after the administering thetherapy; (d) subjecting the at least one first body fluid sample to afirst chromatography-mass spectroscopy analysis to determineconcentration in the at least one first body fluid sample of at leastone oxysterol selected from the group consisting of 7-ketocholesteroland cholestane-3β,5α,6β-triol; (e) subjecting the at least one secondbody fluid sample to a second chromatography-mass spectroscopy analysisto determine concentration in the at least one second body fluid sampleof the at least one oxysterol; and (f) determining magnitude ofdifference between the at least one oxysterol concentration of the atleast one first body fluid sample and the at least one second body fluidsample, whereby a decrease in concentration of the at least oneoxysterol in the second sample compared to the first sample indicatesefficacy of the therapy.
 2. A method in accordance with claim 1, whereinthe therapy is selected from the group consisting of enzyme replacementtherapy, gene therapy, statin administration, administration of acholesterol synthesis inhibitor, administration of a liver X receptor(LXR) agonist, administration of an orphan nuclear receptor PXR ligands,administration of a neurosteroid, administration ofN-butyldeoxynojirimycin (NB-DNJ) (miglustat), administration ofcurcumin, administration of an antibiotic, administration of salsalate,administration of salicylic acid, administration of an RXR ligand,administration of a sphingolipid synthesis inhibitor (myriocin),administration of KCl, administration of EGTA, administration of acalcium channel inhibitor, administration of nifedipine, administrationof verapamil, administration of an antioxidant, administration ofvitamin E, administration of vitamin C, administration ofaurintricarboxylic acid, administration of flavonoids, administration ofan estrogen, administration of propyl gallate, administration ofglutathione, administration of a caspase inhibitor, administration of aMAP kinase inhibitor, administration of a peroxisomeproliferator-activated receptor (PPARs) ligand, administration of15d-PGJ2, administration of WY 14643, administration of indomethacin,administration of a gluccorticoid, administration of dexamethasone,administration of hydrocortisone, administration of a PI-3 kinaseinhibitor, administration of an NMDA open channel blocker, dietarytherapy and a combination thereof.
 3. A method in accordance with claim1, wherein the at least one oxysterol is cholestane-3β,5α,6β-triol.
 4. Amethod in accordance with claim 1, wherein the at least one oxysterol is7-ketocholesterol.
 5. A method in accordance with claim 4, wherein thetherapy is cyclodextrin therapy.
 6. A method in accordance with claim 1,wherein each of the first chromatography-mass spectroscopy analysis andthe second chromatography-mass spectroscopy analysis comprises: (a)adding to the body fluid sample a known amount of an internal standardcomprising an oxysterol other than the at least one oxysterol; (b)isolating the at least one oxysterol from the biological sample; and (c)quantifying the isolated at least one oxysterol.
 7. A method inaccordance with claim 6, wherein the quantifying the at least oneoxysterol comprises determining the relative concentration of the atleast one oxysterol and the known amount of internal standard in thebody fluid sample by correlating the area under the curve obtained forthe at least one oxysterol with the area under the curve obtained forthe internal standard.
 8. A method in accordance with claim 1, whereinthe first biological sample and the second biological sample are eachselected from the group consisting of plasma, serum, blood, sputum andamniotic fluid.
 9. A method in accordance with claim 1, wherein thedisorder involving the accumulation of one or more oxysterols isselected from the group consisting of a lysosomal storage disease, acholesterol trafficking disease, a neurodegenerative disease and acombination thereof.
 10. A method in accordance with claim 1, whereinthe disorder involving the accumulation of one or more oxysterols isselected from the group consisting of Niemann-Pick C (NPC) disease,infantile neuronal ceroid lipofuscinosis, GM1 gangliosidosis (“GM-1”),GM-2 gangliosidosis (“GM-2”), Gaucher's disease and hepatosplenomegaly.11. A method in accordance with claim 1, wherein the disorder involvingthe accumulation of an oxysterol is Niemann-Pick C (NPC) disease.
 12. Amethod for determining efficacy of a therapy in a subject afflicted witha disorder involving the accumulation of one or more oxysterols,comprising: (a) obtaining at least one first body fluid sample from thesubject prior to administering a therapy to the subject; (b)administering the therapy to the subject; (c) obtaining at least onesecond body fluid sample from the subject after the administering thetherapy; (d) subjecting the at least one first body fluid sample to afirst chromatography-mass spectroscopy analysis to determineconcentration in the at least one first body fluid sample of24-hydroxycholesterol; (e) subjecting the at least one second body fluidsample to a second chromatography-mass spectroscopy analysis todetermine concentration in the at least one second body fluid sample of24-hydroxycholesterol; and (f) determining magnitude of differencebetween the 24-hydroxycholesterol concentration of the at least onefirst body fluid sample and the at least one second body fluid sample,whereby an increase in concentration of 24-hydroxycholesterol in thesecond sample compared to the first sample indicates efficacy of thetherapy.
 13. A method in accordance with claim 12, wherein the therapyis selected from the group consisting of enzyme replacement therapy,gene therapy, statin administration, administration of a cholesterolsynthesis inhibitor, administration of a liver X receptor (LXR) agonist,administration of an orphan nuclear receptor PXR ligands, administrationof a neurosteroid, administration of N-butyldeoxynojirimycin (NB-DNJ)(miglustat), administration of curcumin, administration of anantibiotic, administration of salsalate, administration of salicylicacid, administration of an RXR ligand, administration of a sphingolipidsynthesis inhibitor (myriocin), administration of KCl, administration ofEGTA, administration of a calcium channel inhibitor, administration ofnifedipine, administration of verapamil, administration of anantioxidant, administration of vitamin E, administration of vitamin C,administration of aurintricarboxylic acid, administration of flavonoids,administration of an estrogen, administration of propyl gallate,administration of glutathione, administration of a caspase inhibitor,administration of a MAP kinase inhibitor, administration of a peroxisomeproliferator-activated receptor (PPARs) ligand, administration of15d-PGJ2, administration of WY 14643, administration of indomethacin,administration of a glucocorticoid, administration of dexamethasone,administration of hydrocortisone, administration of a PI-3 kinaseinhibitor, administration of an NMDA open channel blocker, dietarytherapy and a combination thereof.
 14. A method in accordance with claim12, wherein each of the first chromatography-mass spectroscopy analysisand the second chromatography-mass spectroscopy analysis comprises: (a)adding to the body fluid sample a known amount of an internal standardcomprising an oxysterol other than 24-hydroxycholesterol; (b) isolating24-hydroxycholesterol from the biological sample; and (c) quantifyingthe isolated 24-hydroxycholesterol.
 15. A method in accordance withclaim 15, wherein the quantifying the 24-hydroxycholesterol comprisesdetermining the relative concentration of the 24-hydroxycholesterol andthe known amount of internal standard in the body fluid sample bycorrelating the area under the curve obtained for the24-hydroxycholesterol with the area under the curve obtained for theinternal standard.
 16. A method in accordance with claim 12, wherein thefirst biological sample and the second biological sample are eachselected from the group consisting of plasma, serum, blood, sputum andamniotic fluid.
 17. A method in accordance with claim 12, wherein thedisorder involving the accumulation of one or more oxysterols isselected from the group consisting of a lysosomal storage disease, acholesterol trafficking disease, a neurodegenerative disease and acombination thereof.
 18. A method in accordance with claim 12, whereinthe disorder involving the accumulation of one or more oxysterols isselected from the group consisting of Niemann-Pick C (NPC) disease,infantile neuronal ceroid lipofuscinosis, GM1 gangliosidosis (“GM-1”),GM-2 gangliosidosis (“GM-2”), Gaucher's disease and hepatosplenomegaly.19. A method in accordance with claim 12, wherein the disorder involvingthe accumulation of an oxysterol is Niemann-Pick C (NPC) disease.
 20. Amethod in accordance with claim 12, wherein the disorder involving theaccumulation of an oxysterol is Tay-Sachs disease.